The hERG potassium channel is a major cause of drug-induced Iong-QT syndrome. Electrophysiology and unnatural amino-acid mutagenesis at key positions will be employed to define binding of drugs to bERG channels expressed in mammalian cells. The drugs to be investigated are chosen from various structural classes: cisapride, terfenadine, dofetilide, astemizole, MK-499, quinidine, vesnarinone, and chloroquine. The unnatural amino acids to be incorporated include series that manipulate cation-pi, hydrophobic, hydrogen-bond, and main-chain interactions. The initial experiments will generate further hypotheses about additional positions, leading to a series of conclusions about the nature of contact points for the unique interaction between each drug and the channel. Furthermore, defective trafficking of mutant bERG potassium channels is a major cause of inherited Iong-QT syndrome. Fluorescent unnatural amino-acid side chains will be used to investigate trafficking of wild type and mutant hERG channels in real time in living mammalian cells. Fluorescence resonance energy transfer (FRET) between hERG subunits will monitor assembly and stoichiometry of the channel and the details of intracellular trafficking;and FRET between hERG and other proteins will define further interactions. Electroporation techniques, novel orthogonal tRNA suppressor species, and levels of elongation and release factors will be optimized for unnatural amino-acid incorporation by nonsense suppression in mammalian cells. These ongoing methodological developments will enhance the studies and will also provide general tools for investigating membrane proteins that contain unnatural amino-acid residues in mammalian cells. This project contributes to progress on the NIH roadmap goals, "Molecular Libraries and Imaging".